Coronary artery disease (CAD) is the leading cause of death in the industrialized world.1 CAD in men and women represents enormously different clinical challenges attributable to a sex variation in symptomatology, risk stratification, and efficacy of therapies.2 Current treatment targets the existing plaque lesion, via angioplasty, stents, and lipid-lowering agents.2 However, promoting repair to inhibit plaque formation could be more optimal for vascular health.

We showed that administration of mononuclear (combined hematopoietic- and stromal-derived) bone marrow (BM) fractions from “young” nonatherosclerotic apolipoprotein-E knockout (apoE−/−) mice or from older C57BL/6 mice attenuated plaque growth in middle-aged apoE−/− mice.3 The BM mononuclear cells (BMNCs) homed to the plaque lesions, suggesting cell-mediated repair of the vessel wall. In apoE−/− mice, a subpopulation of BM CD31+/CD45− vascular progenitor cells (VPCs) diminished with age, which correlated with absence of atheroprotection.3 More recently, higher endothelial progenitor cell (EPC) counts and activity have been inversely correlated with severity of CAD, so that for every increase of 10 EPC colony-forming units, the likelihood of CAD declined by 20%.4 These data suggest that BM EPCs and VPCs may be capable of vascular repair.

The impact of donor or recipient sex on BMNC-mediated atheroprotection is unknown. Burke et al showed a higher predominance of plaque erosion in younger women,5 which suggests that atheroprotection may have sex-based differences. We investigated the role of sex on vascular repair by examining differences in the capacity of exogenous BMNCs to reduce plaque formation in a sex-matched and -mismatched apoE−/− mice fed a high-fat diet.

Materials and Methods

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org. A schematic of the experimental protocol is illustrated in Figure 1.

Definition of Progenitor Cell Populations

Because there are variations in the definitions of progenitor cell populations in the literature, in this study, we a priori defined BM-derived EPCs as AC133+/CD34+ cells, VPCs as CD31+/CD45low cells, and the BM inflammatory cell–containing fraction as the CD45+ subpopulation. BMNCs injected into male and female apoE−/− recipients were derived from C57BL6/J mice (10±2 weeks of age) and maintained in vitro for 48 hours, as described in the online data supplement.

Statistical Analysis

A complete description of statistical analysis procedures appears in the online data supplement. In all analyses, statistical significance was set at P≤0.05.

In both sexes, reductions in BM-CD34+ cells occurred as atherosclerosis progressed (Figure 2B). In males, percentage cell counts fell in parallel with plaque growth. In females, the reduction of the BM-CD34+ cell percentage occurred 7 weeks prior to the highest plaque burden accumulation; the decline in cell counts was smaller than in male mice of a similar (21 weeks) age (P=0.01). By 32 weeks of age, the BM-CD34+ cell percentages were similar in both sexes.

In male apoE−/− mice, BM-VPCs demonstrated a continuous linear fall as plaque accumulated in the vessels (Figure 2C). On the contrary, in females, VPC percentage counts were stable through week 21, even though atherosclerosis progressed, but as plaque burden further increased, BM-VPCs fell significantly.

In both males and females, BM-EPCs exhibited a distinct pattern compared with other BM progenitors (Figure 2D). In contrast to depletion of CD34+ and VPCs in parallel to plaque buildup, BM-EPCs rose significantly at 21 weeks but then ultimately fell as the atherosclerotic process continued. In female mice, where plaque accumulated significantly slower through week 21 compared with males, BM-EPC percentage counts were higher (P<0.001); conversely, the BM-EPCs declined less at 32 weeks than in male mice (P=0.01), even though female atherosclerotic plaque burden was significantly higher.

After donor BMNCs were maintained in vitro for 48 hours, relative EPC number increased, with females exhibiting significantly more EPCs than males (11.1±1.4% [n=3] vs 7.4±1.3% [n=3]; P=0.04). However, VPC percentage fell in both sexes (females: from 19.8±4.9% to 5.8±0.6%, n=3; males: from 18.6±3.5% to 4.1±2.3%, n=3; both P=0.01, paired t test). Similar to EPCs, the VPC percentages were significantly higher in samples derived from female donors (P=0.04).

When BMNCs were administered to apoE−/− recipients, a significant reduction in aortic plaque burden was observed in male mice that received female donor BMNCs compared with vehicle-treated and male donor BMNCs (Figure 3A and 3B). No significant changes in plaque burden were observed in female apoE−/− recipients irrespective of donor sex (Figure 3B).

Sex and Effects of BMNC Treatment

Exogenous Bone Marrow Progenitors Alter Recipient Bone Marrow

The relative percentages of CD34+ cells, EPCs, VPCs, and CD45+ cells in the BM of recipients changed after BMNC administration in a sex-dependent fashion. In male recipients, treatment with BMNCs of either sex boosted the percentage of CD34+ cells (Figure 3C) and EPCs (Figure 3F) in BM to levels similar to those in treated or untreated females (all P=NS). These equal increases after BMNC therapy occurred despite the differences in the percentages of EPCs in male and female donor samples. In female recipients, the treatment did not change percentages of BM-CD34+ or BM-EPC cells.

The inflammatory cell–containing BM-CD45+ fraction significantly increased only in male apoE−/− mice treated with female BMNCs (P=0.04; Figure 3E): the group of animals that exhibited attenuation of atherosclerotic plaque formation. BM-CD45+ counts were unchanged in all other animals.

In a multivariate regression analysis, higher percentage counts of BM-EPCs and the CD45+ cell fraction preserved correlation with lesser plaque burden. However, the univariate association of BM-CD34+ cells with reduced plaque was lost (Table 1).

Female mice were not subjected to univariate or multivariate analyses because of the absence of significant differences in plaque burden after BMNC administration (Figure 3B).

Changes in Cytokines/Chemokines With BMNC Treatment

After donor BMNCs were maintained in vitro for 48 hours, expression of T-helper (Th)1-type (proinflammatory) chemokine tumor necrosis factor (TNF)-α was similar in the media from cells of either sex, but Th2-type (anti-inflammatory) cytokines interleukin (IL)-4 and IL-5, and the pleiotropic/regulatory cytokine IL-6 were present in 1.9-to 3.0-fold higher concentrations in female cell media versus male cell or cell-free media (data not shown). Granulocyte colony-stimulating factor (G-CSF) and IL-15 were detected only in female BMNC media, whereas male BMNC and cell-free media concentrations were equivalent (G-CSF: 23.9±8.4 pg/mL in female BMNCs and 6.3±5.8 pg/mL in male BMNCs, P=0.02; IL-15: 34.0±35.2 pg/mL in female BMNCs and undetected in male BMNCs; n=3 each). None of the other 16 cytokines was detected.

BMNC treatment created an inflammatory response in apoE−/− mice; the type and the magnitude of the effect varied by sex. In male apoE−/− mice, treatment with either male or female BMNCs increased IL-1β, IL-12, TNF-α, and Regulated on Activation, Normal T-Cell Expressed and Secreted, reflecting a Th1-type proinflammatory response (Table I in the online data supplement). Increases of these cytokines/chemokines were numerically larger in male mice treated with female BMNCs: the group of animals that showed attenuation of plaque formation. Along with the increase in Th1-type mediators, Th2-type cytokines (IL-5, IL-10, IL-13, and, to a lesser degree, monocyte chemoattractant protein-1) levels rose as well; larger numerical increases in Th2-type cytokines occurred in male mice with reduced plaque burden. Upregulation of Th1- and Th2-type cytokines paralleled the increase in BM-CD45+ cell fraction in these mice (Figure 3E). In female apoE−/− mice, fewer Th1- and more Th2-related cytokines were upregulated after treatment with either male or female BMNCs (supplemental Table I) but without atheroprotection (Figure 3B).

Median G-CSF levels were higher in vehicle-treated females compared with males and did not increase after either male or female BMNCs (supplemental Table I). G-CSF and IL-15 (but not KC) moderately correlated in vehicle-treated females (but not males) and also in female apoE−/− mice after treatment with female BMNCs (supplemental Figure I).

Discussion

In this study, we have shown that BMNC-mediated atheroprotection differs in apoE−/− male and female mice fed a high-fat diet and that the difference may be, in part, related to varying degrees of endogenous repair already underway in animals of each sex and supplemented by differences in the response to male- and female-derived BMNC populations. Atheroprotective capabilities of BMNCs were observed only when cells from wild-type female mice were administered to atherosclerotic apoE−/− males. In the context of the natural history of murine atherosclerosis, female BMNCs reduced plaque burden by ≈40%. Of note, a recent large clinical trial of CAD patients demonstrated only a 7% reduction in plaque volume with rosuvastatin.7 Therefore, atheroprotection with BMNCs holds an enormous promise, especially considering that 16 million Americans suffer from CAD1 and that 1 of every 2.7 deaths is attributed to atherosclerosis.1 A considerable amount of work needs to be done before BMNC treatment of CAD becomes a clinical reality. In addition to discerning the mechanism of atheroprotection, we need to understand why the benefit in male recipients was confined to female BMNCs and why female recipients appeared indifferent to BMNCs.

We attempted to dissect the sex-based differences in the progression of atherosclerosis and the changes in the relative numbers of endogenous BM progenitor cells. In addition, we sought to begin to understand the mechanistic impact of exogenous BMNC administration after repair by assessing 22 circulating cytokine/chemokines. Our data offered some understanding of the interactions that mediate vascular repair while uncovering notable sex differences. Specifically, the speed of progression of atherosclerosis and the amount of plaque accumulated differed in male and female apoE−/− mice. Changes in BM progenitors accompanied disease progression in a sex-dependent fashion, which suggested differences in endogenous repair.

Male and female animals responded differently to BMNCs at the level of the BM and the vessel. BMNCs administered to male and female apoE−/− mice boosted BM-EPC counts, ≈2-fold, to levels indistinguishable from either treated or untreated females. Yet female donor BMNCs contained significantly more EPCs, and their delivery attenuated plaque formation. This result suggests that rather than simply traveling to the BM, the “excess” EPCs participated in vascular repair. Our multivariate analysis, which showed the correlation of reduced plaque burden with the percentages of EPCs delivered, supports this supposition. Combined with our previous observations that exogenous cells travel to the sites of injury,3 the data from this study suggest that BMNCs act locally as well as at the level of BM. However, the outcome of treating male apoE−/− mice with male BMNCs, where BM-EPCs increased but plaque formation was not attenuated, suggests that the BM effect alone is insufficient for repair. Therefore, the dose of EPCs delivered may be a critical component of success of atheroprotection. If so, reducing plaque with male BMNCs may be as simple as administering a higher percentage of EPCs. However, male animals that received female cells exhibited attenuation of plaque formation. Because the sex-based difference in the in vitro and in vivo hormonal and inflammatory milieu may be responsible for the differences in the reparative capacity of exogenous cells, infusing higher numerical counts of EPCs may not suffice. Dose–response studies combined with in vitro functional assays of male and female cells are required to clarify whether female EPCs are truly more “reparative” or whether the number of EPCs delivered determines atheroprotection.

In treated animals, an increase in “inflammation” was registered, together with a lower plaque burden. Specifically, upregulation of CD45+ cell–containing BM fraction (which contains precursors of granulocytes, monocytes, B cells, etc)8 correlated with repair. Even though inflammation is observed with progression of atherosclerosis,9,10 in our study, it was associated with repair. As indicated previously,3 we propose that initial inflammation is a “positive” signal to trigger recruitment of endogenous BM progenitors. When cells capable of repair are recruited in sufficient numbers, repair ensues and inflammation decreases. If insufficient numbers of and/or cells functionally incapable of repair are recruited, the repair process stops and inflammation increases and becomes a “negative” signal to the detriment of the tissue. Our data, as well as that of 2 recent studies, support this hypothesis. Veillard et al11 demonstrate that inflammation surges between weeks 4 and 10 of high-fat diet in apoE−/− mice, but then acute phase response transforms into chronic inflammation as atherosclerosis progresses. Inoue et al12 provide evidence that local arterial inflammation serves as a signal for the release of BM progenitors after stent deployment to promote the healing cascade within the vessel.

Increased G-CSF levels correlated with the reparative response. In addition, hematopoietic cytokines IL-15 and KC (IL-8) correlated with the G-CSF increase. IL-8 has been shown to mediate progenitor cells migration.13 IL-15 stimulates proliferation of dendritic T cells,14 and there is evidence of regulatory CD4+CD25+ T-cell engagement by IL-15.15 Caux et al have shown that dendritic T cells are derived from CD34+ progenitors in presence of TNF-α.16 Our animals exhibited increased BM-CD34+ cells and circulating TNF-α, together with reduced plaque burden. Therefore, it is tempting to hypothesize that 2 processes are required for attenuation of plaque: (1) inflammation, to mobilize the necessary BM progenitors and to trigger digestion of lesions by macrophages; and (2) vessel repair, which includes engraftment of EPCs to renew the activated endothelium, and a decrease in smooth muscle activation and in attachment of lipid particles.17 Both processes have been shown to involve G-CSF.18 Higher G-CSF levels in vehicle-treated female mice, where plaque growth was stable, support its role in endogenous repair. Whether G-CSF is the conductor of the repair process and other cytokines/chemokines are the orchestra remains to be confirmed. Although G-CSF reduced left ventricular remodeling after acute myocardial infarction,19 clinical data of filgrastim (G-CSF) administration in CAD patients have been mixed, and untoward effects, including restenosis and de novo lesions, have been reported.20,21 Exacerbation of atherosclerosis in apoE−/− mice fed a high-fat diet after G-CSF treatment (10 μg/kg per day for 5 days, every other week for 4 weeks)22 puts forward the question of an optimal regimen to produce comparable (with BMNCs) vascular repair.

The significantly higher G-CSF and exclusive IL-15 production by female (versus male) BMNC fraction in vitro reaffirms our idea that attenuation of plaque is the result of actions of the infused BMNCs, but the secondary activation of endogenous repair via mobilization of BM progenitors and concomitant Th1-Th2 shift is also necessary for repair. The cytokines/chemokines measured in the media of cultured BMNCs differed from the published data on those produced by CD34+ cells.23 Specifically, TNF-α, IL-1β, Regulated on Activation, Normal T-Cell Expressed and Secreted, and IL-8 were secreted by CD34+ cells. These differences in the inflammatory milieu suggest that administration of CD34+ cells versus BMNCs may activate different pathways, which, in turn, may beget dissimilar clinical outcomes. However, age (ie, functional capacity and available number of cells) may influence the reparative capacity of BMNCs, CD34+ cells, and EPCs. We have shown previously atheroprotection with BM mononuclear fractions derived from young but not older atherosclerotic apoE−/− mice.3

Female recipient mice responded completely differently to exogenous BMNCs from males. Even though circulating cytokines/chemokines were elevated and BM-VPCs increased (versus males), BM-CD34+ cells and BM-EPCs remained stable and so did plaque burden. Although the exact mechanism(s) of lack of plaque attenuation are unknown, our data let us propose several possibilities. First, the females were treated earlier in the course of atherosclerosis (prior to the maximal increase in plaque burden) compared with males. Secondly, at the time of treatment, BM-VPCs counts did not decline proportionately to plaque (as they did in males), which together with higher BM-CD34+ counts and upregulated G-CSF in vehicle-treated mice suggests a better ongoing endogenous repair than in males.3 In addition, females exhibited a stronger Th2-type response following BMNCs than males. Of note, Pinderski et al showed inhibition of atherosclerosis in LDL receptor–deficient mice that had IL-10–overexpressing T cells.24 Finally, failure to increase BM-EPCs despite receiving a relatively high dose of EPCs (in BMNC samples) implies a regulatory mechanism. Therefore, exogenous BMNCs were placed in an environment in which endogenous repair was more efficient (than in males) and were thus extraneous, so that no additional atheroprotection could be seen. This finding itself may have clinical implications and suggests that cell delivery should be timed with need. In other words, females may benefit from BMNC administration significantly later than males.

Sex-mismatched hematopoietic stem cell transplantation in humans may cause acute graft-versus-host reaction (GVHD), in which donor T-cells attack the minor histocompatibility (Y-chromosome) antigens of the host, causing interferon-γ– and IL-2–driven cytokine storm, along with antibody production by the host to reject transplanted cells.25 If GVHD were to occur, atherosclerosis would be exacerbated.25 On the contrary, atheroprotection occurred in male apoE−/− mice that received female BMNCs. Similarly, a lack of significant increase in plaque growth in females that received male cells favors the absence of GVHD.

Plaque attenuation occurred without reduction of hypercholesterolemia. In recent years, the core understanding of atherosclerosis has shifted away from the exclusive engagement with hyperlipidemia to a long-term relationship with inflammation.10,17 In fact, male BMNCs reduced total cholesterol in recipients of both sexes but without atheroprotection. Although this finding was unanticipated, recent evidence suggests the existence of cholesteryl ester transfer protein in BM,26 which could partially explain the reduction in total cholesterol but does not account for failure to lessen plaque burden.

Lastly, estrogen has been shown to influence BM progenitor cell–based myocardial repair after acute myocardial infarction.27,28 In our study, even though administration of sex-matched or -mismatched BMNCs increased estriol levels, there was no direct relationship of estriol levels and atheroprotection. Although we acknowledge the limitation of the pooled data, we also recognize that more insight into the role of estrogen could have been obtained if estrus cycle data were collected and related to atheroprotection. Estrus cycle may modulate reparative activity of BMNCs, as recently shown by Masuda et al.29

In summary, the present study provides a first step toward defining and deciphering sex differences in vascular repair. Although apoE−/− mice have a significantly greater hyperlipidemia than do CAD patients,6 it is evident that atherosclerosis begets different inflammatory milieus in males and females. Likewise, endogenous repair and response to cell therapy differs between the sexes. These differences may account for some of the sex disparities in plaque morphology seen by pathologists and for differences in symptomatology and efficacy of therapies between men and women seen by clinical cardiologists.

Conclusion

We have shown a sex difference in response to BMNC therapy for atheroprotection. Administration of BMNCs derived from wild-type female donors attenuated plaque formation in only male atherosclerotic apoE−/− mice. The percentages of EPCs and CD45+ cells in the BM of recipients, as well as G-CSF levels, significantly correlated with a lower plaque burden. Male BMNCs administered to males and BMNCs from donors of either sex infused into females showed no reduction in plaque. In males, Th1- and Th2-type cytokines increased with BMNC therapy and increases in these cytokines correlated with plaque attenuation. In contrast, females exhibited a stronger Th2-type response following administration of BMNC of either sex. Endogenous BM progenitor cell populations changed with atherosclerosis: CD34+ cells and VPCs decreased, but EPCs rose and then fell as plaque accumulated.

Overall, our findings may have implications for clinical cell therapy trials for CAD, because men and women may exhibit differential responses to exogenous BMNCs. Further exploration of sex differences in vessel repair is warranted.

Acknowledgments

This work was supported in part by the Medtronic Foundation and by funding from the Center for Cardiovascular Repair, University of Minnesota. W.D.N. was supported in part by NIH T32 grant AR007612 (program director, David D. Thomas, PhD).